Evidence of epithelial remodelling but not epithelial–mesenchymal transition by transcriptome profiling in vernal keratoconjunctivitis
Authors: Andrea Leonardi, Philippe Daull, Umberto Rosani, Fabiano Cavarzeran, Elena Salami, Jean‐Sebastien Garrigue, Brun Paola
Journal: Allergy (2022)

Vernal keratoconjunctivitis (VKC) is a severe type 2 ocular disease associated with corneal involvement, tissue remodelling, potential complications and visual impairment.1 The tarsal (T-VKC), limbal (L-VKC) and mixed (M-VKC) phenotypes are characterized by either the presence of tarsal giant papillae, infiltrates/papillae/nodules at the limbus or by the coexistence of both, respectively. The clinical research has been focused on the identification of inflammatory mediators to untangle the immune mechanisms involved and to identify potential targets for therapeutic interventions. Recently, we showed different gene expression between the three different phenotypes, with modulations of genes involved in innate and adaptive immunity, antigen presentation, Th2- and Th17-priming.2 Furthermore, our data suggest that epithelial barrier dysfunction and the epithelial-to-mesenchymal transition (EMT) may play a role in the VKC pathogenesis.3 Aiming to identify differences in gene expression between VKC and normal subject (CT) with a particular focus to epithelial and EMT-related genes, we obtained impression cytology samples (Eyeprim™ device, OPIA Technologies SAS) from 15 active VKC patients (Table S1) and five healthy age-matched CT. The VKC severity was evaluated using three different clinical scores (Appendix S1). The study complied with the tenets of the Declaration of Helsinki and was approved by the IRB of our Institution. Informed consent was obtained from the subjects (or patients), who were informed of the nature of the study. The GeneChip™ WT Pico Kit (Thermofisher Scientific) has been used to analyse the modulation of the transcriptome based on 21,448 probes, revealing 325 DEGs comparing VKC to CT: 92 upregulated and 241 downregulated (Figure 1A). Considering the three different phenotypes, 17 differently expressed genes (DEGs) were identified in T-VKC, 232 in L-VKC and 166 in M-VKC, with few common DEGs (Figure 1B). The regression analysis based on clinical scores and performed to identify genes whose expression associates with disease severity, showed that 397, 461 and 297 DEGs were identified in patients with high Bonini's, CLEK and Oxford scores, respectively (Table S2). Enrichment analyses showed that in T-VKC several gene ontology biological process (GOBP) were related to the tissue remodelling and B cells regulations and cytokine production (Table S3), while in L-VKC, the ribonucleoprotein complex biogenesis was the most represented GOBP. By Reactome analysis, comparing with CT, 169 out of 325 identifiers were found in all VKC cohort, and 128 out of 226 in L-VKC (Table S4). Many of the genes involved in these pathways belong to the ribosomal and mitochondrial ribosomal proteins families suggesting that further investigations are needed to relate environmental stress, typical of VKC, unfolded or misfolded proteins and ribosomal functions. Since EyePrim collects mostly epithelial cells, we looked at the expression of genes related to the epithelial barrier function. Of the many genes, either up- or down-regulated (Table 1), TJP1, K23 (validated by IHC, as shown in Figure S1) and GJB2 were upregulated, CLDN8 and GNGT1 were highly downregulated similarly to what reported for atopic dermatitis4 and Sjogren syndrome,5 suggesting a possible epithelial barrier dysfunction and remodelling in VKC. Furthermore, because of the potential evolution in fibrosis, we found an upregulation of epithelial marker E-cadherin, a non-increased expression of genes encoding for EMT markers, and a downregulation of the transcription factors required for EMT (Table S5), suggesting that EMT is inhibited in VKC besides TGF-β, a potent inducer of both remodelling and EMT, is upregulated, as previously reported.6 In conclusion, different VKC phenotypes and severities reveal different trends of gene expression, highlighting the role of the epithelial barrier in the complexity of the disease pathogenesis. Further analyses, possibly based on whole transcriptome methods, will certainly expand our knowledge on molecular mechanisms during VKC and on possible endotypes that underline the different VKC phenotypes. None. Open Access Funding provided by Universita degli Studi di Padova within the CRUI-CARE Agreement. Open Access Funding provided by Universita degli Studi di Padova within the CRUI-CARE Agreement. This study was supported by an unrestricted grant from Santen SAS. The authors have no conflict of interest, only Philippe Daull and Jean-Sébastien Garrigue are the employees of Santen SAS. Appendix S1 Figure S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.